Lipid absorption and overall intestinal lymphatic transport are impaired following partial small bowel resection in mice

Short bowel syndrome (SBS) is associated with diminished levels of serum fats caused by unknown mechanisms. We have shown that mesenteric lymphatics remodel to a more primitive state one week after small bowel resection (SBR); therefore, this study focuses on the effect of chronic lymphatic remodeling and magnitude of resection on intestinal lipid uptake and transport. C57BL6 and Prox1 creER-Rosa26LSLTdTomato (lymphatic reporter) mice underwent 50% or 75% proximal SBR or sham operations. Functional transport of lipids and fecal fat content was measured and lymphatic vasculature was compared via imaging. There was a significant reduction in functional transport of cholesterol and triglyceride after SBR with increasing loss of bowel, mirrored by a progressive increase in fecal fat content. We also describe significant morphological changes in the lymphatic vasculature in both the lamina propria and mesentery. Intestinal lymphatic drainage assay in vivo demonstrated a marked reduction of systemic absorption after resection. Intestinal lymphatic vessels significantly remodel in the setting of chronic SBS. This remodeling may account at least in part for impaired intestinal uptake and transport of fat via the compromised lymphatic architecture. We believe that these changes may contribute to the development of intestinal failure associated liver disease (IFALD), a major morbidity in patients with SBS.


Results
Malabsorption of cholesterol and triglycerides after SBR. At an early time point after resection, postoperative day 7, the distal intestine reprograms to a more proximal identity with an upregulation of genes associated with lipid handling and metabolism 16 . In agreement with this reprogramming, we observed a progressive increase in expression of ApoB in the proximal intestine and MTTP in the remaining distal intestinal segment, which regulate the assembly of chylomicrons, in the resected mice (Fig. 1A, B; ANOVA p < 0.05 and p < 0.01, respectively). As we have previously shown in single-cell analysis at early time points after resection, there was an increase in the intestinal lipid-sensing gene FABP2 in the distal intestine ( Fig. 1B; ANOVA p < 0.005) 16 . Simultaneously, in the distal intestine, there was a decrease in the long-chain fatty acid transporter gene, CD36, the cholesterol transporter gene, Abca1, and the bile acid binding gene, FABP6, expression in the distal intestine with increasing loss of bowel ( Fig. 1B; ANOVA p < 0.005, p < 0.005, and p < 0.05, respectively). These data show that the intestine reprograms to promote lipid absorption and chylomicron assembly, leading us to test whether fat absorption normalized in concert with this reprogramming.
Despite the favorable programming of genes to orient toward fat absorption, with increasing loss of bowel, total serum cholesterol decreased with 75% resected mice having a 67% decrease compared to non-operated controls ( Fig. 2A; ANOVA p < 0.0001). Serum campesterol, a marker of dietary cholesterol absorption, followed this same trend of decreased concentrations with increased extent of bowel resection ( Fig. 2B; ANOVA p < 0.001). Lathosterol, an intermediate in cholesterol synthesis, also decreased in the serum as proportion of intestine resected increased ( Fig. 2C; ANOVA p < 0.05). However, as the lathosterol to campesterol ratio increased with the magnitude of resection (Fig. 2D), the extent of malabsorption appears greater than the decrease in cholesterol synthesis. To directly assess absorption of exogenous cholesterol, we performed a time-course of serum fluorescence following oral delivery of the fluorescently labeled cholesterol mimetic TOPFLUOR cholesterol. With increasing loss of bowel, there was a decreasing amount of TOPFLUOR cholesterol absorbed (Fig. 2E, F; ANOVA p < 0.0001).
These data pointed to failed absorption of fat, leading to seek other ways to confirm this observation. Failed absorption of dietary fat would be expected to result from increased fat content in feces. At one year after SBR, with increasing loss of bowel after resection, fecal fat concomitantly increased (Fig. 3A, B; ANOVA p < 0.0005), a feature that held true when the data were normalized to average caloric intake ( Fig. 3C; ANOVA p < 0.05). There was no difference in fat to lean mass ratios across the groups (Fig. 3D). To assess chylomicron transport via lymphatics, we performed a functional assay of lipid absorption using C16-Bodipy-labeled olive oil (C16-Bodipy is a neutral lipid that binds to olive oil upon mixture). This showed a decrease in chylomicron absorption with a lipid load over time in the resected mice (Fig. 3E, F; ANOVA p < 0.0001). Serum free fatty acids at 12-15 weeks after resection also followed this same trend ( Fig. 3G; ANOVA p = 0.0007). , sham (n = 5), 50% SBR (n = 5), and 75% SBR (n = 4) mice. *p < 0.05, **p < 0.005. www.nature.com/scientificreports/ These data indicate that failed intestinal lipid absorption is present in mice long after surgical resection. Overall, we conclude that fat malabsorption exists as a chronic consequence of SBR. SBR promoted a 51% reduction in serum bile acids after an oral fat load, which may result in reduced secretion of bile acids in the intestinal lumen to promote lipid uptake (Fig. 4A, B; p < 0.05). However, as an additional mechanism, there may also be a role for lymphatic remodeling, which we next set out to explore. The data so far reveal reduced food intake of mice with 75% SBR and higher fecal fat. Yet, absorption defects were similar between mice with 50% versus 75% SBR. Thus, to proceed with fewer confounding factors, we continued our studies focusing on the lymphatic vasculature in mice receiving 50% SBR.

Scientific
Intestinal mucosal lymphatic capillaries change after chronic SBR. We have previously shown dilation of the upstream mucosal lymphatic capillaries in the remnant distal bowel seven days after resection 15 , but it was unclear if over time the remodeled lymphatics returned to their baseline arrangement or remained remodeled. We thus extended the period that we assessed the lymphatic vasculature to 3-4 months following surgical resection. In cross-sectional images of the small intestine (Fig. 5A), we observed the previously documented 40% increase in villus height by post-operative week 12-15 ( Fig. 5B; p < 0.0001), serving as a positive control for adaptive remodeling. Lymphatic capillary luminal area in the lamina propria was significantly increased in the distal intestine by 66% and 70% in resected mice compared to sham and intraoperative controls, www.nature.com/scientificreports/ respectively; there was also a pattern of increasing lymphatic area in the proximal intestine ( Fig. 5C; p < 0.05, p < 0.005). Image analysis for the number lymphatic capillary vessels by both the total length and the total lamina propria area showed no change in the proximal intestine but significantly decreased in the 50% resected mice compared to both intraoperative and sham controls in the distal intestine (Fig. 5D, E).
Mesenteric collecting lymphatic vessel changes after chronic SBR. In addition to the alterations observed in the distal small intestine lymphatic mucosal capillary network, we examined the effect of resec- www.nature.com/scientificreports/  www.nature.com/scientificreports/ tion on the mesenteric lymphatic vessels. At postoperative week 13, collecting lymphatic vessels present in the mesentery and emerging from the intestinal wall distal to the anastomosis still showed the substantially altered morphology following 50% SBR versus sham control mice ( Fig. 6A) that we had earlier observed at day 7 15 . The resected mice had an average 31% increase in mesenteric branch width compared to sham controls ( Fig. 6B; p < 0.05). Furthermore, resected mice had an average 31% increase in lymphatic budding area into the mesenteric sheath compared to sham controls ( Fig. 6C; p < 0.05).
Compromise of lymphatic flow after intestinal resection. The findings above collectively revealed that lymphatic remodeling persisted chronically after SBR. We thus wondered if lymph transport might be altered at these time points. To test the intestinal lymphatic drainage function in SBR, we performed a standardized micro injection of a fluorescent marker into Peyer's patches in vivo with timed measurements via serum fluorescence. This experiment tests that capacity of the lymphatic system to transport soluble cargo from the intestine in the lymph, of which chylomicrons are a major component. Gut that drains to the downstream mesenteric lymphatic network showed compromised flow in resected mice compared to controls (Fig. 7A, B; ANOVA p < 0.05). Accumulation of the tracer in the draining lymph node was 78% and 70% reduction in mice receiving surgical removal of 50% of the small bowel compared to nonoperative and sham controls, respectively ( Fig. 7C; p < 0.005). These data reveal that marked impairment of lymphatic transport accompanies the altered morphology and may account at least in part for the failed absorption of lipid nutrients.

Discussion
Here, we show by analysis of fecal fat content and by tracing the fate of orally delivered fluorescent cholesterol analogue that dietary lipid absorption is impaired after experimental SBR many weeks beyond the point of surgical resection of the small bowel. Furthermore, we found a significant reduction in the blood cholesterol metabolite campesterol in our mice. This reduction resembles a similar trend in the plasma of SBS patients that have been studied after withdrawal from parenteral nutrition 14 . Because campesterol arrives to plasma via absorption from the gut, the finding further supported the conclusion of impaired absorption or transport, although reduction in diet-derived campesterol might in part be due to partially reduced food intake in mice receiving 75% SBR. All nutrient absorption is compromised in SBS patients due to the loss of absorptive surface area, lipid absorption is considered to be the most vulnerable, in part due to compromise of the enterohepatic circulation, decreased bile acid pool, and decreased pancreatic lipase secretion [17][18][19] . After establishing evidence for failed lipid absorption persisting after SBR in mice, the question became what mechanisms are causal. Failed transport of fats may indicate impaired duodenal and jejunal lymph transport, as lipid absorption is most prominent in these sites. Alternatively or additionally, impaired lipid transport might www.nature.com/scientificreports/ point toward reduced capacity of the gut to take up or package fats. The bile acid pool was reduced, and indeed may contribute importantly to reduced lipid uptake. In addition, our findings suggest that the lymphatic vasculature was also impaired. Besides morphological changes in the lymphatic vasculature after SBR, fluorescent dextran tracer deposited in the submucosa-draining lymphatic network of the intestine confirmed significantly impaired lymph flow using an approach without confounding possible changes in absorption. One week after resection, early lymphatic remodeling in the intestinal mucosal lymphatics is characterized by reduced mucosal lymphatic capillary vessel area 15 . Interestingly, after a prolonged period of resection, we have shown that lymphatic capillary area actually increases compared to both intraoperative and sham controls in the distal intestine. We believe that is likely secondary obstruction of flow in the mesentery causing upstream dilation in the intestinal mucosa. We also witnessed this in the mesenteric collecting vessels as the branch width was also dilated after resection. Similar to prior observations seen at POD7, resected mice also had an increase in lymphatic budding area into its mesenteric sheath compared to controls. This budding is normally observed during early development in mice 20,21 .
In addition to possibly direct effects on lipid absorption along the epithelium, could failed lymphatic transport impact lipid absorption such that lipid remained luminal in the intestine and ultimately appeared in the feces? While the idea that altered lymphatic function might impact absorption across the epithelium may seem counterintuitive, there is strong precedent for it. Preceding studies revealed that loss of lymphatic integrity due to altered signaling in VEGFR3 within lymphatic lacteals leads to increased fecal fat load 22,23 . Furthermore, conditions that lead to the closure of intercellular junctions in the lacteals also prevents dietary fat uptake and diverts it to the feces 24 .
The causal root of lymphatic changes after small bowel resection is likely multifactorial, with chronic disturbance of flow downstream of the surgery likely having a major effect on lymphatic phenotype and likely resulting in prolonged inflammation. However, the mechanisms of impaired lymph transport remain unknown. After massive intestinal resection, there is a critical, programmed compensatory response in the epithelium resulting in both adaptation (with increased enterocyte proliferation) and apoptosis 25 . Indeed, an emerging concept is www.nature.com/scientificreports/ that intestinal epithelial cells and the underlying lymphatic vessels coordinately communicate 26,27 . While it is demonstrated that the status of the lymphatic vasculature affects the integrity of intestinal epithelial cells, whether a major change in the status of the epithelium affects the lymphatic vasculature has not been studied. Perhaps future manipulations to improve the status of epithelial cells may improve the lymphatic vasculature or vice versa. Overall, we have shown that the early lymphatic remodeling structural changes occur chronically after SBR and contribute to functionally compromised lipid absorption. Further studies are necessary to determine if other lymphatic cargo, such as vitamins and immune cells, are also affected after resection. Perturbations in lymphatic function after SBR likely also contribute to greater delivery of luminal endotoxin into the portal vein, thereby contributing to liver injury 28 . Indeed, we have found severe liver injury and fibrosis in resected mice at 15 weeks following SBR when placed on a higher fat diet 29 . Liver injury and fibrosis are the most lethal clinical consequences of SBS 30 . Manipulations of enteral fat may therefore be a critical therapeutic strategy to prevent this significant morbidity.
These data indicate that intestinal lymphatic vessels significantly remodel in the setting of chronic short bowel syndrome. This remodeling results in impaired intestinal transport of fat via the compromised lymphatic architecture, contributing to decreased fatty acid uptake. We believe that these changes may contribute to the development of IFALD, a major morbidity in patients with SBS.

Materials and methods
Animals. As described previously, C57BL/6 J 9-12 week-old male mice were obtained from Jackson Laboratories (Bar Harbor, ME) 15  Operations and harvest. Mice underwent either a sham control operation, 50% proximal SBR, or a 75% proximal SBR, as previously described 33,34 . There was also a cohort of mice that did not undergo an operation (non-op), but were treated identically. Mice were placed on liquid diet for 12-24 h preoperatively to minimize the risk of anastomotic obstruction. In brief, for 50% and 75% SBRs, bowel was first exteriorized via a midline laparotomy and then transected 1 to 2 cm distal to the ligament of Treitz and 12 cm or 6 cm proximal to the ileocecal junction, respectively. An end-to-end anastomosis was handsewn with interrupted 9-0 nylon sutures. Sham operations involved a transection with re-anastomosis 12 cm distal to the ileocecal junction. Intraoperative (IO) resected distal bowel was fixed in 10% formalin and embedded in paraffin for immunohistochemistry and immunofluorescence. Postoperatively, mice were kept in an incubator and fasted for 12-24 h before resuming the liquid diet.
RNA extraction and quantitative reverse transcription-polymerase chain reaction. As described previously, total RNA from proximal and distal intestinal tissue from C57BL/6 J mice harvested approximately 1.5 years postoperatively was isolated using RNeasy Mini kits per the manufacturer's protocol (Qiagen, Germantown, MD). Proximal intestine was approximately 6 cm harvested from 2 cm past the gastroduodenal junction and distal intestine was the 6 cm leading up to the ileocecal junction, so as to consistently take the same bowel from all operative types (non-operative, sham, and SBRs) 29 . qRT-PCR was conducted using the ABI StepOnePlus Real- Serum free fatty acid, cholesterol, lathesterol, and campesterol levels. Serum free fatty acid, and cholesterol levels were measured using a commercially available kit (Wako Chemicals, Richmond, VA) on C57BL/6 J at 12-15 weeks after SBR. Serum lathesterol and campesterol levels were measured on C57BL/6 J mice 12-13 weeks after SBR. The lathosterol and campesterol esters in mouse plasma were hydrolyzed with potassium hydroxide, and total lathosterol and campesterol were extracted with liquid-liquid extraction in the presence of d7-lathosterol and d6-campesterol as internal standards for lathosterol and campesterol, respectively. The plasma samples and 8-point calibration samples were derivatized with nicotinic acid to improve the mass spectrometric sensitivity. The lathosterol and campesterol were separated by reversed phase liquid chromatography and detected with positive multiple-reaction monitoring on an Applied Biosystems Sciex 4000QTRAP tandem mass spectrometer. The peak integration and construction of standard curve were performed with Analyst 1.6.3. The calibration curve was constructed by plotting the peak area ratios of analyte to internal standard versus the www.nature.com/scientificreports/ corresponding concentrations using 1/x 2 weighted least square regression. Serum free fatty acid, and cholesterol levels were performed on mice with confirmed adaptation; adaptation was not assessed in mice tested for serum campesterol and lathosterol levels.

Functional cholesterol transport.
Intestinal uptake of cholesterol was measured using TopFluor-cholesterol (Avanti Polar Lipids, Alabaster, AL) in C57BL/6 J mice at post-operative week 31 35,36 . Mice were fasted for six hours during the mouse dark cycle and then gavaged with TopFluor (8 μg/gm body weight) with methylβ-cyclodextrin (1 mg/gm body weight; Sigma-Aldrich, St. Louis, MO). Tail vein serum was measured at time points 0, 1, 2, 4, and 6 h after gavage. Methyl-β-cyclodextrin is given with TopFluor in order to deplete already present plasma membrane cholesterol 37,38 . Serum fluorescence was determined using Cytation 5 multiplate reader (Biotek, Winooski, VT). Mice were excluded if fluorescence curve was not achieved secondary to a technical gavage error resulting in post-gavage emesis and incomplete distribution of gavage bolus.
Food consumption and fecal fat content. Food consumption and fecal fat content were analyzed on mice aged approximately one year after operation. After acclimation to individual metabolic cages, food intake was measured and feces were collected for two days. As previously described, fecal fat content was determined gravimetrically 39 . In brief, dried feces (0.2gm) was solubilized overnight in 1.6 mL water and extracted in 5 mL chloroform:methanol (2:1). The organic phase was transferred into a pre-weighed vial and the homogenate was re-extracted again with 2 mL of cholorform:methanol (2:1). The organic phase was transferred again and, in total, was dried under nitrogen and reweighed to determine the lipid mass. Percent fat absorption was determined by normalizing the lipid mass to food consumption. Immunohistochemistry and immunofluorescence. As described previously, intestinal tissue from 50% SBR and sham control mice 1 cm distal to their anastomosis was harvested 12 weeks post-operatively, fixed in 10% formalin and embedded in paraffin similar to the IO samples taken from the distal bowel resected in the 50% SBR mice at operation 15 . Additionally, proximal gut was obtained from both sham and 50% SBR mice 15 weeks after operation at approximately 5 cm from the gastro-duodenal junction. Longitudinal, 5-μm thick sections were created and hematoxylin and eosin stained for villus height measurement to asses for structural adaptation (NIS elements AR 4; Nikon, Melville, NY), which was set at > 15% increase from IO samples 41 . For immunofluorescence, slides were deparaffinized and antigen retrieval (Diva Decloaking solution, Biocare Medical, Concord, CA) was performed under pressure for 10 min. Slides were blocked in donkey serum (5%, Sigma-Aldrich), bovine serum albumin (1%, Sigma-Aldrich), and Triton-X100 (0.03%, Pharmacia Biotech). Slides were incubated overnight at 4 °C with primary antibodies diluted in 0.2% bovine serum albumin and then secondary antibodies were added for 1 h. Antibodies used include rabbit anti-mouse Lymphatic vessel endothelial hyaluronic acid receptor (LYVE-1; 1:600, Abcam, ab14917), Cy3-conjugated donkey anti-rabbit IgG (1:400, Jackson ImmunoResearch, 711-165-152) and FITC-conjugated mouse anti-mouse alpha-smooth muscle actin IgG (1:500, Sigma, F3777, Clone #1A4). A confocal microscope (Leica SPE and SP8) was used to capture images and blinded analysis was performed using Imaris (Bitplane, Switzerland) and FIJI (ImageJ) software (National Institute of Health, Bethesda, MD) of at least 6 mm of intestine for each sample. LYVE-1 + , which is a marker of lymphatic endothelial cells, vessel structures present in the lamina propria were defined as mucosal lymphatics and slides were excluded if staining was of poor quality 42 .

Measurement of body composition.
Whole mount mesenteric imaging. Mesentery from Prox1-Cre-ER T2 xRosa26 LSL tdTomato mice who were both post-operative week 13 from 50% SBR and sham control operations and also postoperative 1.5 years from 75% SBR and sham control operations was pinned and fixed in 4% paraformaldehyde + 30% sucrose for at least 12 h and then transferred into phosphate-buffered saline, as described previously 15 . The expression of Prospero homeobox protein 1 (PROX1), which governs lymphangiogenesis, is restricted to lymphatic endothelial cells 43 . Fluorescent imaging of the lymphatic branches distal to the anastomosis was then performed (Leica M205FA). Blinded image analysis, including average branch width and area of budding structures was per- www.nature.com/scientificreports/ formed using Imaris (Bitplane, Switzerland) and FIJI (ImageJ) software (National Institute of Health, USA) on the postoperative week 13 50% SBR and sham samples.

Functional lymphatic flow.
In mice 12-14 weeks after SBR, anesthetized mice were placed in a custombuilt stage to perform intravital recordings using our fluorescence stereoscope, as described previously 44 . Physiological conditions were maintained by a temperature-controlled metal stage with 3 temperature sensors (Dual Channel Temperature Controller, Warner Instruments, TC-344C), controlling the temperature independently at 3 sites at ~ 36-37 °C. Intestine and mesentery were placed over a SYLGARD ® 184 (Dow Corning, Nidland, MI) stage and securely attached with fine pins (Fine Science Tools, 26002-20). A midline incision of the peritoneum was used to expose the cecum and terminal ileum, along with the mesentery and mesLNs. Mesentery and intestine were kept moist and under controlled temperature with buffer using a peristaltic pump and In-Line Solution Heater (Harvard Apparatus, 64-0102). To address the intestinal lymphatic draining function, we injected ~ 1.5 µL of 2000 kDa FITC-Dextran (Sigma-Aldrich, FD2000S, or Invitrogen, D7137) into a Peyer's patch in the ileum, which was the closest to the anastomosis able to be reached in sham and resected mice and the third from the cecum in non-operative mice. After, serum fluorescence, obtained via tail vein, was determined using Cytation 5 multiplate reader (Biotek, Winooski, VT) at 0, 5, 10, 15, 30, 45, and 60 min after the Peyer's patch injection and presented as fold change from baseline.
Statistical analysis. Statistical analysis was performed GraphPad-Prism 6 software (La Jolla, CA). Levels of triglyceride, free fatty acid, cholesterol, campesterol, lathosterol, TopFluor cholesterol, C16-Bodipy, fecal fat, mRNA expression levels, and bile acid levels, were analyzed using a one-way ANOVA with Tukey's multiple comparisons between groups. Intestinal adaptation (increases in villus height) and lymphatic network characteristics were analyzed using the unpaired Student's t test. A p value of < 0.05 was considered significant. Data are expressed as the mean + / − SEM with significance of multiple comparison analyses displayed.